Intestinal epithelium is composed of a monolayer of morphologically polarized cells, which function in absorbing substances from the intestine and transporting them ultimately to the bloodstream. Several epithelial in vitro cell culture systems have been established in the past few decades, which mimic the cells lining the intestine. Some of the cell lines are Caco-2, HT-29, SW 1116, T84, IEC-18, and IEC-6. Caco-2 and HT-29 intestinal cell lines are the most widely used and best characterized systems. Other experimental models that have been under investigation include various clones of these lines (e.g., Caco-2/TC-7 (Caro et al., Int. J. Pharm., 1995, 116), Caco-2BBe (Peterson and Mooseker, J. Cell Sci., 1992 102(3)), HT29-MTX (Pontier et al., J. Pharm. Sci., 2001, 90(10)), co-culturing systems (e.g., Caco-2/HT29-MTX(Walter et al., J. Pharm. Sci., 1996, 85(10)), and primary cell cultures (Fukamachi, J. Cell Sci., 1992, 103).
The cell lines have been an invaluable aid in studies of drug transport, microbial infection, and enzyme induction/regulation. For the most part, Caco-2 cells are the cells of choice for drug transport research and analysis. These cells differentiate spontaneously in culture some time after reaching confluence. Caco-2 cells usually require about 14-30 days after reaching confluence, with an average of 21 days post-confluence, to fully differentiate, i.e. differentiate to the point at which they exhibit many of the morphological, enzymatic, and most importantly, the nutrient transport characteristics of normal human intestinal cells. This is a comparatively long time to wait for cells to be ready to perform experiments, and requires thoughtful long-term planning for conducting studies with this system.
Attempts have been made to decrease the time required for development and differentiation of a Caco-2 cell monolayer, as well as other differentiated epithelial cell cultures. Most of the approaches involve the addition of various differentiation and growth factors to the culture medium, the use of a collagen support for cell attachment, and the reduction of serum requirements in the media. Currently, the 3-day BIOCOAT® HTS Caco-2 assay model (Becton Dickinson Laboratory) is the only commercially available system, and uses fibrillar collagen as cell substrate and serum-free medium. The Biocoat® system allows establishment of a differentiated enterocyte monolayer within three days in a serum-free environment. In this system, the cells are seeded on the Biocoat® Fibrillar Collagen Cell Culture Inserts (Becton Dickinson) and cultured in the specialized medium supplemented with butyric acid, hormones, growth factors and other defined metabolites. The assessment of the Biocoat® and other accelerated systems, such as the one developed by Lentz et al. (Lentz et al., Pharm. Sci., 1998, 1:S456), revealed that these rapid models allow one to determine the rank order of permeability of compounds and give results equivalent to those in the traditional 21-day culture system (Liang et al., J. Pharm. Sci., 2000, 89(3)). However, some of the previously developed accelerated Caco-2 systems have been shown to express low P-glycoprotein (P-gp) levels and have leaky tight junctions. (Liang et al., J. Pharm. Sci., 2000, 89(3)). While the BIOCOAT® system does not exhibit such problems, the use of this commercially available model is quite costly for routine experimental work, because the successful high-throughput culturing of the accelerated Caco-2 system requires frequent purchasing of the specialized media and Fibrillar Collagen Cell Culture Inserts. Moreover, it is unknown whether the collagen coating interferes with drug transport, and thus not provide a true picture of drug absorption, and comparison to the traditional model could be a problem. Therefore, there is a need to develop an inexpensive, non-collagen dependent, rapid Caco-2 model that could be used routinely for high throughput screening of potential drugs.